Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990); Lewis et al., in Chemistry of the Amazon, Biodiversity Natural Products, and Environmental Issues, 588, (P. R. Seidl, O. R. Gottlieb and M. A. C. Kaplan) 135-167, ACS Symposium Series, Washington D.C. (1995)). Because of their pronounced antibiotic properties (Markkanen, T. et al., Drugs Exptl. Clin. Res. 7:711-718 (1981)), antioxidant properties (Fauré, M. et al., Phytochemistry 29:3773-3775 (1990); Osawa, T. et al., Agric. Biol. Chem. 49:3351-3352 (1985)) and antifeedant properties (Harmatha, J., and Nawrot, J., Biochem. Syst. Ecol. 12:95-98 (1984)), a major role of lignans in vascular plants is to help confer resistance against various opportunistic biological pathogens and predators. Lignans have also been proposed as cytokinins (Binns, A. N. et al., Proc. Natl. Acad Sci. USA 84:980-984 (1987)) and as intermediates in lignification (Rahman, M. M. A. et al., Phytochemistry 29:1861-1866 (1990)), suggesting a critical role in plant growth and development. It is widely held that elaboration of biochemical pathways to lignins/lignans and related substances from phenylalanine (tyrosine) was essential for the successful transition of aquatic plants to their vascular dry-land counterparts (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products. Evolution and Function, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, D.C. (1994)), some four hundred and eighty million years ago (Graham, L. E., Origin of Land Plants, John Wiley & Sons, Inc., New York, N.Y. (1993)).
Based on existing chemotaxonomic data, lignans are present in “primitive” plants, such as the fern Blechnum orientale (Wada, H. et al., Chem. Pharm. Bull. 40:2099-2101 (1992)) and the hornworts, e. g., Dendroceros japonicusand Megaceros flagellaris (Takeda, R. et al., in Bryophytes. Their Chemistry and Chemical Taxonomy, Vol. 29 (Zinsmeister, H. D. and Mues, R. eds) pp. 201-207, Oxford University Press: New York, N.Y. (1990); Takeda, R. et al., Tetrahedron Lett. 31:4159-4162 (1990)), with the latter recently being classified as originating in the Silurian period (Graham, L. E., J. Plant Res. 109: 241-252 (1996)). Interestingly, evolution of both gymnosperms and angiosperms was accompanied by major changes in the structural complexity and oxidative modifications of the lignans (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products. Evolution and Function, 562 (W. D. Nes, ed. ) 202-246, ACS Symposium Series: Washington, D.C. (1994); Gottlieb, O. R., and Yoshida, M., in Natural Products of Woody Plants. Chemicals Extraneous to the Lignocellulosic Cell Wall (Rowe, J. W. and Kirk, C. H. eds.) pp. 439-511, Springer Verlag: Berlin (1989)). Indeed, in some species, such as Western Red Cedar (Thuja plicata), lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.
In addition to their functions in plants, lignans also have important pharmacological roles. For example, podophyllotoxin, as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990)). Antiviral properties have also been reported for selected lignans. For example, (−)-arctigenin (Schröder, H. C. et al., Z. Naturforsch 45c, 1215-1221 (1990)), (−)-trachelogenin (Schröder, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)) and nordihydroguaiaretic acid (Gnabre, J. N. et al., Proc. Natl. Acad Sci. USA 92:11239-11243 (1995)) are each effective against HIV due to their pronounced reverse transcriptase inhibitory activities. Some lignans, e. g., matairesinol (Nikaido, T. et al., Chem. Pharm. Bull. 29:3586-3592 (1981)), inhibit cAMP-phosphodiesterase, whereas others enhance cardiovascular activity, e. g., syringaresinol β-D-glucoside (Nishibe, S. et al., Chem. Pharm. Bull. 38:1763-1765 (1990)). There is also a high correlation between the presence, in the diet, of the “mammalian” lignans or “phytoestrogens”, enterolactone and enterodiol, formed following digestion of high fiber diets, and reduced incidence rates of breast and prostate cancers (so-called chemoprevention) (Axelson, M., and Setchell, K. D. R, FEBS Lett. 123:337-342 (1981); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 41:3-8 (1992); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 52:97-103 (1995)). The “mammalian lignans,” in turn, are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al., J. Applied Bacteriol., 58:37-43 (1985)).
The biosynthetic pathways to the lignans are only now being defined. Based on radiolabeling experiments with crude enzyme extracts from Forsythia intermedia, it was first established that entry into the 8,8′-linked lignans, which represent the most prevalent dilignol linkage known (Davin, L. B., and Lewis, N. G., in Rec. Adv. Phytochemistry, Vol. 26 (Stafford, H. A., and Ibrahim, R. K., eds), pp. 325-375, Plenum Press, New York, N.Y. (1992)), occurs via stereoselective coupling of two achiral coniferyl alcohol molecules, in the form of oxygenated free radicals, to afford the furofuran lignan (+)-pinoresinol (Davin, L. B., Bedgar, D. L., Katayama, T., and Lewis, N. G., Phytochemistry 31:3869-3874 (1992); Paré, P. W. et al., Tetrahedron Lett. 35:4731-4734 (1994)).
Recently, the initial step in the 8-8′ linked lignan biosynthetic pathway was clarified in F. intermedia (Davin, L. B., Wang, H.-B., Crowell, A. L., Bedgar, D. L., Martin, D. M., Sarkanen, S., Lewis, N. G., Science 275:362-366 (1997)). This involved stereoselective monolignol coupling of two molecules of coniferyl alcohol in the presence of a 78 kDa dirigent protein and a one-electron oxidase (such as laccase). The one-electron oxidant is considered only to provide oxidative capacity, with the dirigent protein binding, orientating, and coupling the free-radical forms and releasing (+)-pinoresinol. The dirigent protein was purified from F. intermedia stem tissue and its encoding gene cloned (Gang, D. R., Costa, M. A., Fujita, M., Dinkova-Kostova, A. T., Wang, H. B., Burlat, V., Martin, W., Sarkanen, S., Davin, L. B., Lewis, N. G., Chemistry & Biology 6:143-151(1999)).
In Forsythia intermedia, and presumably other species, (+)-pinoresinol undergoes sequential reduction to generate (+)-lariciresinol and then (−)-secoisolariciresinol (Katayama, T. et al., Phytochemistry 32:581-591 (1993); Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)). The reductions catalyzed by pinoresinol/lariciresinol reductase proceed via abstraction of the pro-R hydride of NADPH, resulting in an “inversion” of configuration at both the C-7 and C-7′ positions of the products, (+)-lariciresinol and (−)-secoisolariciresinol (Chu, A., et al., J. Biol. Chem. 268:27026-27033 (1993)). Pinoresinol/lariciresinol reductase was purified ˜3200 fold to apparent electrophoretic homogeneity from a soluble crude protein extract; this was achieved by employing a series of affinity, hydrophobic interaction, hydroxyapatite, gel filtration, and ion exchange chromatographic steps (Dinkova-Kostova, A. T., Gang, D. R., Davin, L. B., Bedgar, D. L., Chu, A., Lewis, N. G., J. Biol. Chem. 271:29473-29482 (1996)). The purified protein was demonstrated to be a type A NADPH-dependent reductase.
The corresponding pinoresinol/lariciresinol reductase gene (called plr-Fi1) was cloned from a Forsythia cDNA library (Dinkova-Kostova, A. T., Gang, D. R., Davin, L. B., Bedgar, D. L., Chu, A., Lewis, N. G., J. Biol. Chem. 271:29473-29482 (1996)), and its fully functional recombinant protein then over-expressed in E. coli using a pET-based expression system (pSBETa vector) (Schenk, P. M., Baumann, S., Mattes, R., Steinbiβ, H.-H., BioTechniques 19:196-200 (1995)). It was found that the only products formed following incubation of the recombinant pinoresinol/lariciresinol reductase with (±)-pinoresinols in the presence of NADPH were (+)-lariciresinol and (−)-secoisolariciresinol, i. e., only (+)-pinoresinol and (+)-lariciresinol, and not (−)-pinoresinol nor (−)-lariciresinol, served as substrates. Thus, the recombinant enzyme catalyzed exactly the same enantiospecific conversion as for the native plant protein from Forsythia (Dinkova-Kostova, A. T., Gang, D. R., Davin, L. B., Bedgar, D. L., Chu, A., Lewis, N. G., J. Biol. Chem. 271:29473-29482 (1996); Lewis, N. G., Davin, L. B., in: Comprehensive Natural Products Chemistry, Vol. 1. (Barton, Sir D. H. R., Nakanishi, K., and Meth-Cohn, O., eds), pp 639-712, Elsevier, London (1999)). (−)-Matairesinol is subsequently formed via dehydrogenation of (−)-secoisolariciresinol, further metabolism of which presumably affords lignans such as the antiviral (−)-trachelogenin in Ipomoea cairica and (−)-podophyllotoxin in Podophyllum peltatum. 
Thus, the stereospecific formation of (+)-pinoresinol and the subsequent reductive steps giving (+)-lariciresinol and (−)-secoisolariciresinol are pivotal points in lignan metabolism, since they represent entry into the furano, dibenzylbutane, dibenzylbutyrolactone and aryltetrahydronaphthalene lignan subclasses. Additionally, it should be noted that while lignans are normally optically active, the particular enantiomer present may differ between plant species. For example, (−)-pinoresinol occurs in Xanthoxylum ailanthoides (Ishii et al., Yakugaku Zasshi, 103:279-292 (1983)), and (−)-lariciresinol is present in Daphne tangutica (Lin-Gen, et al., Planta Medica, 45:172-176 (1982)). The optical activity of a particular lignan may have important ramifications regarding biological activity. For example, (−)-trachelogenin inhibits the in vitro replication of HIV-1, whereas its (+)-enantiomer is much less effective (Schroder et al., Naturforsch. 45c:1215-1221(1990)).
The lignan, matairesinol, is an important component of the plant arsenal that helps confer dietary benefits to humans, specifically against the onset of breast and prostate cancers (Adlercruetz, H. and Mazur, W. Anal. Med., 1997, 29:95-120). This lignan is found in various whole-grain cereal food, seed and berries, and is converted by intestinal bacteria to form enterolactone; the latter compound is considered to be the primary metabolite in conferring the health protection. Additionally, the lignan, matairesinol, also has an important function in conferring quality, color and durability to specific heartwoods, such as the highly valued western red cedar (Thuja plicata) species via its conversion into plicatic acid and its congeners. Using Forsythia intermedia as a model system, it was established that matairesinol is formed in planta via dehydrogenation of secoisolariciresinol (FIG. 1) (Umezawa, T., Davin, L. B. and Lewis, N. G., Biochem. Biophys. Res. Commun., 1990, 171(3), 1008-1014; Umezawa, T., Davin, L. B., Kingston, D. G. I., Yamamoto, E. and Lewis, N. G., J. Chem. Soc., Chem. Commun., 1990, 1405-1408; Umezawa, T., Davin, L. B. and Lewis, N. G., J. Biol. Chem., 1991, 266:10210-10217).